Project Summary/Abstract The goal of this project is to develop a tissue engineering technology applicable to stem cell-based therapies for Alzheimer's disease (AD). AD is the most common dementia and characterized by synaptic dysfunction and neuronal loss. There is currently no effective cure for the disease. Transplantation of human neural stem cells (hNSCs) to replace lost neurons is a promising strategy. However, the cells transplanted into the AD brain would face a toxic environment with elevated levels of the amyloid-? (A?) peptides, which would induce synaptic damage and cell death for implanted neurons. How can we overcome this problem? We previously identified the activity of synaptic protein takusan (Tu et al., 2007). Overexpression of takusan in rat primary neuronal cultures leads to enhancement of various synaptic properties including dendritic-spine density, and AMPA- and NMDA-mediated glutamate receptor activity. Interestingly, these are the synaptic properties that are attenuated by A? exposure. We then overexpressed takusan in cultured rat neurons and challenged them by soluble A? oligomers. Remarkably, takusan expression mitigated synaptic loss in these neurons. By deletion mapping and domain swapping, we invented the synthetic 69-aa long protein hD2b, in which we fused the human D2 takusan domain derived from discs-large-homolog 5 (DLG5) and a human PDZ-binding C-terminal sequence. Overexpression of hD2b in cultured rat neurons, like that of full-length takusan variants, conferred protection against oA?-induced synaptic toxicity. In the current proposal, we will produce hNSCs expressing hD2b and test whether this genetic modification is beneficial toward the development of a novel cell-replacement therapy against AD. This proposal allows us to leverage our expertise in stem cell technology at Allele Biotech Inc. with the knowledge and invention of hD2b protein by our academic collaborators. Specific Aims are as follows: Aim 1. To produce human neurons (hNs) that express hD2b in vitro. Aim 2. To test whether hNs expressing hD2b are more resistant against oA?-induced synaptic toxicity compared to control hNs.. Aim 3. To evaluate whether transplantation of hNSCs expressing hD2b into the brain of human amyloid precursor protein (hAPP) transgenic mice, hAPP-J20, will rescue their behavioral and histological phenotypes. This proposal consists of experiments that have been performed, reagents tested, and conclusions published in series of high quality papers, using rat primary neurons. Since the proposed work is a logical extension to human neurons, we have high confidence that positive results will be obtained through a 1 year phase 1 SBIR effort. Upon completion of phase 1, we expect to continue to phase 2 projects to perform additional animal testing aimed at near-future IND and eventual partnership with pharmaceutical companies.